Currently used antiretrovirals targeting the viral reverse transcriptase, protease, and envelope proteins have reduced mortality and morbidity in HIV-infected patients. However, the emergence of viral strains resistant to the inhibitors, and the appearance of side effects after prolonged drug administration pose important problems to the efficacy of HAART treatments. Characterization and evaluation of novel targets for therapeutic intervention is a priority in HIV/AIDS research. CD4 down-modulation plays an important role in HIV pathogenesis. In vitro, failure to down-modulate CD4 results in the production of HIV-1 particles with severely reduced infectivity whereas in vivo enhanced receptor down-modulation confers a replicative advantage to HIV-1 in primary lymphocytes. To date, no specific inhibitors of this function have been characterized. To facilitate the discovery of new drugs, we propose to develop a fusion-based assay to screen for specific inhibitors of the HIV-induced CD4 down-modulation activity. This assay will rely on the fact that the rate of fusion between gp120-expressing cells and CD4-positive cells can be modulated by the concentration of these proteins on the cell surface. We hypothesize that since HIV down-modulates expression of CD4, interfering with this function in infected cells will result in higher amounts of surface-CD4, and therefore fusion will occur to a higher extent than in cells in which efficient down-modulation occurs. Experiments to evaluate the sensitivity and specificity of this assay are proposed in Specific Aim 1. To validate the assay, we will screen synthetic combinatorial libraries for potential inhibitors of the virus-induced CD4 down-modulation (Specific Aim 2). We hypothesize that drugs inhibiting this function will impair HIV replication. Our goal is to identify lead compounds with defined chemical structures that could evolve into a discovery program for a new class of anti-HIV drugs. The specific aims are: Aim 1, to develop a fusion-based assay for the identification of inhibitors of the HIV-induced CD4 down-modulation, and; Aim 2, to characterize small-molecule inhibitors of the HIV-induced CD4 down-modulation activity.